Genome-wide loss-of-function genetic screen identifies INSIG2 as the vulnerability of hepatitis B virus-integrated hepatoma cells.
CDK2
CRISPR/Cas
HBV
liver cancer
pooled library screen
Journal
Cancer science
ISSN: 1349-7006
Titre abrégé: Cancer Sci
Pays: England
ID NLM: 101168776
Informations de publication
Date de publication:
29 Jan 2024
29 Jan 2024
Historique:
revised:
20
12
2023
received:
09
10
2023
accepted:
29
12
2023
medline:
30
1
2024
pubmed:
30
1
2024
entrez:
30
1
2024
Statut:
aheadofprint
Résumé
There are approximately 250 million people chronically infected with hepatitis B virus (HBV) worldwide. Although HBV is often integrated into the host genome and promotes hepatocarcinogenesis, vulnerability of HBV integration in liver cancer cells has not been clarified. The aim of our study is to identify vulnerability factors for HBV-associated hepatocarcinoma. Loss-of-function screening was undertaken in HepG2 and HBV-integrated HepG2.2.15 cells expressing SpCas9 using a pooled genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) library. Genes whose guide RNA (gRNA) abundance significantly decreased in HepG2.2.15 cells but not in HepG2 cells were extracted using the MAGeCK algorithm. We identified four genes (BCL2L1, VPS37A, INSIG2, and CFLAR) that showed significant reductions of gRNA abundance and thus potentially involved in the vulnerability of HBV-integrated cancer cells. Among them, siRNA-mediated mRNA inhibition or CRISPR-mediated genetic deletion of INSIG2 significantly impaired cell proliferation in HepG2.2.15 cells but not in HepG2 cells. Its inhibitory effect was alleviated by cotransfection of siRNAs targeting HBV. INSIG2 inhibition suppressed the pathways related to cell cycle and DNA replication, downregulated cyclin-dependent kinase 2 (CDK2) levels, and delayed the G
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : Japan Agency for Medical Research and Development
ID : JP23ama221410
Organisme : Japan Agency for Medical Research and Development
ID : JP23fk0210110
Organisme : Japan Agency for Medical Research and Development
ID : JP23fk0210131
Organisme : Japan Agency for Medical Research and Development
ID : JP23fk0310501
Organisme : Japan Agency for Medical Research and Development
ID : JP23fk0310512
Organisme : Japan Agency for Medical Research and Development
ID : JP23fk0310524
Informations de copyright
© 2024 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.
Références
Trepo C, Chan HLY, Lok A. Hepatitis B virus infection. Lancet. 2014;384:2053-2063.
Organization WH. Guidelines for the prevention, care and treatment of persons with chronic hepatitis B infection. 2015.
Jiang Y, Han QJ, Zhao HJ, Zhang J. The mechanisms of HBV-induced hepatocellular carcinoma. J Hepatocell Carcinoma. 2021;8:435-450.
Peneau C, Imbeaud S, La Bella T, et al. Hepatitis B virus integrations promote local and distant oncogenic driver alterations in hepatocellular carcinoma. Gut. 2022;71:616-626.
Llovet JM, Kelley RK, Villanueva A, et al. Hepatocellular carcinoma. Nat Rev Dis Primers. 2021;7:6
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816-821.
Murai K, Kodama T, Hikita H, et al. Inhibition of nonhomologous end joining-mediated DNA repair enhances anti-HBV CRISPR therapy. Hepatol Commun. 2022;6:2474-2487.
Koike-Yusa H, Li YL, Tan EP, Velasco-Herrera MD, Yusa K. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat Biotechnol. 2014;32:267-273.
Tzelepis K, Koike-Yusa H, De Braekeleer E, et al. A CRISPR dropout screen identifies genetic vulnerabilities and therapeutic targets in acute myeloid leukemia. Cell Rep. 2016;17:1193-1205.
Kodama M, Kodama T, Newberg JY, et al. In vivo loss-of-function screens identify KPNB1 as a new druggable oncogene in epithelial ovarian cancer. Proc Natl Acad Sci U S A. 2017;114:E7301-E7310.
Kodama T, Suemura S, Myojin Y, et al. Crispr loss-of-function screen identifies the hippo SIGNALLING pathway AS the mediator of regorafenib efficacy in hepatocellular carcinoma. Hepatology. 2019;70:1183A-1184A.
Sells MA, Chen ML, Acs G. Production of hepatitis-B virus-particles in Hep-G2 cells transfected with cloned hepatitis-B virus-DNA. Proc Natl Acad Sci U S A. 1987;84:1005-1009.
Guo YC, Wang J, Benedict B, et al. Targeting CDC7 potentiates ATR-CHK1 signaling inhibition through induction of DNA replication stress in liver cancer. Genome Med. 2021;13:166.
Li W, Xu H, Xiao TF, et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 2014;15:554.
Konishi M, Wu CH, Wu GY. Inhibition of HBV replication by siRNA in a stable HBV-producing cell line. Hepatology. 2003;38:842-850.
Javanbakht H, Mueller H, Walther J, et al. Liver-targeted anti-HBV single-stranded oligonucleotides with locked nucleic acid potently reduce HBV gene expression in vivo. Mol Ther Nucleic Acids. 2018;11:441-454.
Yamai T, Hikita H, Fukuoka M, et al. SIRT1 enhances hepatitis virus B transcription independent of hepatic autophagy. Biochem Biophys Res Commun. 2020;527:64-70.
Lee AR, Lim KH, Park ES, et al. Multiple functions of cellular FLIP are essential for replication of hepatitis B virus. J Virol. 2018;92:e00339-18.
Zeng H, Qin H, Liao M, et al. CD36 promotes de novo lipogenesis in hepatocytes through INSIG2-dependent SREBP1 processing. Mol Metab. 2022;57:57.
Brooks EE, Gray NS, Joly A, et al. CVT-313, a specific and potent inhibitor of CDK2 that prevents neointimal proliferation. J Biol Chem. 1997;272:29207-29211.
Lei YM, Xu X, Liu HL, et al. HBx induces hepatocellular carcinogenesis through ARRB1-mediated autophagy to drive the G(1)/S cycle. Autophagy. 2021;17:4423-4441.
Mukherji A, Janbandhu VC, Kumar V. HBx-dependent cell cycle deregulation involves interaction with cyclin E/A-cdk2 complex and destabilization of p27Kip1. Biochem J. 2007;401:247-256.
Setton J, Zinda M, Riaz N, et al. Synthetic lethality in cancer therapeutics: the next generation. Cancer Discov. 2021;11:1626-1635.
Kim KH, Seong BL. Pro-apoptotic function of HBV X protein is mediated by interaction with c-FLIP and enhancement of death-inducing signal. EMBO J. 2003;22:2104-2116.
Kurata M, Yamamoto K, Moriarity BS, Kitagawa M, Largaespada DA. CRISPR/Cas9 library screening for drug target discovery. J Hum Genet. 2018;63:179-186.
Wei L, Lee D, Law CT, et al. Genome-wide CRISPR/Cas9 library screening identified PHGDH as a critical driver for sorafenib resistance in HCC. Nat Commun. 2019;10:10.
Jin HJ, Shi YP, Lv YY, et al. EGFR activation limits the response of liver cancer to lenvatinib. Nature. 2021;595:730-734.
Xu F, Tong M, Tong CSW, et al. A combinatorial CRISPR-Cas9 screen identifies Ifenprodil as an adjunct to sorafenib for liver cancer treatment. Cancer Res. 2021;81:6219-6232.
Yang CX, Lee D, Zhang MS, et al. Genome-wide CRISPR/Cas9 library screening revealed dietary restriction of glutamine in combination with inhibition of pyruvate metabolism as effective liver cancer treatment. Advanced. Science. 2022;9:9.
Bao MHR, Yang CX, Tse APW, et al. Genome-wide CRISPR-Cas9 knockout library screening identified PTPMT1 in cardiolipin synthesis is crucial to survival in hypoxia in liver cancer. Cell Rep. 2021;34:34.
Liang JB, Zhao H, Diplas BH, et al. Genome-wide CRISPR-Cas9 screen reveals selective vulnerability of ATRX−mutant cancers to WEE1 inhibition. Cancer Res. 2020;80:510-523.
Yabe D, Brown MS, Goldstein JL. Insig-2, a second endoplasmic reticulum protein that binds SCAP and blocks export of sterol regulatory element-binding proteins. Proc Natl Acad Sci U S A. 2002;99:12753-12758.
Kim JY, Wang LQ, Sladky VC, et al. PIDDosome-SCAP crosstalk controls high-fructose-diet-dependent transition from simple steatosis to steatohepatitis. Cell Metab. 2022;34:1548-1560.e6.
Li WY, Cui XF, Huo Q, et al. Profile of HBV integration in the plasma DNA of hepatocellular carcinoma patients. Curr Genomics. 2019;20:61-68.
Vandenheuvel S, Harlow E. Distinct roles for cyclin-dependent kinases in cell-cycle control. Science. 1993;262:2050-2054.
Malumbres M, Barbacid M. Cell cycle, CDKs and cancer: a changing paradigm. Nat Rev Cancer. 2009;9:153-166.
Goldstein JL, DeBose-Boyd RA, Brown MS. Protein sensors for membrane sterols. Cell. 2006;124:35-46.
Benn J, Schneider RJ. Hepatitis-B virus HBX protein deregulates cell-cycle checkpoint controls. Proc Natl Acad Sci U S A. 1995;92:11215-11219.
Wang X, Huo BN, Liu J, Huang X, Zhang SY, Feng T. Hepatitis B virus X reduces hepatocyte apoptosis and promotes cell cycle progression through the Akt/mTOR pathway in vivo. Gene. 2019;691:87-95.
Ueda H, Ullrich SJ, Gangemi JD, et al. Functional inactivation but not structural mutation of p53 causes liver cancer. Nat Genet. 1995;9:41-47.